JCM Accepts, published online ahead of print on 28 October 2009

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J. Clin. Microbiol. doi:10.1128/JCM.01463-09
Copyright (c) 2009, American Society for Microbiology and/or the Listed Authors/Institutions. All Rights Reserved.

Rapid detection of Mycobacterium tuberculosis and rifampin-resistance using on-demand, near patient technology.

Danica Helb, Martin Jones, Elizabeth Story, Catharina Boehme, Ellen Wallace, Ken Ho, JoAnn Kop, Michelle R. Owens, Richard Rodgers, Padmapriya Banada, Hassan Safi, Robert Blakemore, N. T. Ngoc Lan, Edward C. Jones-López, Michael Levi, Michele Burday, Irene Ayakaka, Roy D. Mugerwa, Bill McMillan, Emily Winn-Deen, Lee Christel, Peter Dailey, Mark D. Perkins, David H. Persing, and David Alland^*

Department of Medicine, New Jersey Medical School – UMDNJ, Newark, NJ; Cepheid, Sunnyvale, CA; Foundation for Innovative New Diagnostics, Geneva, Switzerland; Pham Ngoc Thach Hospital, Ho Chi Minh City, Vietnam; Montefiore Medical Center, Bronx, NY; Department of Pathology, New Jersey Medical School – UMDNJ, Newark, NJ; Makerere University - UMDNJ Research Collaboration, Kampala, Uganda; Department of Medicine, Makerere University School of Medicine, Kampala, Uganda

^* To whom correspondence should be addressed. Email: allandda{at}umdnj.edu.

Current nucleic acid amplification methods to detect Mycobacterium tuberculosis are complex, labor-intensive and technically-challenging. We developed and performed the first analysis of the Cepheid Xpert MTB/RIF Assay, an integrated hands-free sputum-processing and real-time PCR system with rapid on-demand, near-patient technology, to simultaneously detect M. tuberculosis and rifampin-resistance. Analytic tests of M. tuberculosis DNA demonstrated a limit of detection (LOD) of 4.5 genomes per reaction. Studies using sputum spiked with known numbers of M. tuberculosis colony forming units (cfu) predicted a clinical LOD of 131 cfu/mL. Killing studies showed that the assay's buffer decreased M. tuberculosis viability by at least 8 logs, substantially reducing biohazards. Tests of 23 different commonly-occurring rifampin-resistance mutations demonstrated that all 23 (100%) would be identified as rifampin-resistant. An analysis of 20 non-tuberculosis mycobacteria species confirmed high assay specificity. A small clinical validation study of 107 clinical sputum samples from suspected tuberculosis cases in Vietnam detected 29/29 (100%) smear-positive culture-positive cases, and 22/39 (84.6%) or 38/53 (71.7%) smear-negative culture-positive cases, as determined by growth on solid media or both solid and liquid media, respectively. M. tuberculosis was not detected in 25/25 (100%) of the culture-negative samples. A study of 64 smear-positive culture-positive sputa from retreatment tuberculosis cases in Uganda detected 63/64 (98.4%) culture-positive cases and 9/9 (100%) cases of rifampin-resistance. Rifampin-resistance was excluded in 54/55 (98.2%) susceptible cases. Specificity rose to 100% after correcting for a conventional susceptibility test error. In conclusion, this highly-sensitive and simple-to-use system can detect M. tuberculosis directly from sputum in under two hours.